Molecules 2014, 19, 6851-6862; doi:10.3390/molecules19056851 OPEN ACCESS
molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article
Associations of nm23H1, VEGF-C, and VEGF-3 Receptor in Human Prostate Cancer Zui-Su Yang, Yin-Feng Xu, Fang-Fang Huang and Guo-Fang Ding * Engineering Research Centers of Marine Organism Medical Products, Medical College of Zhejiang Ocean University, Zhoushan 316022, China * Author to whom correspondence should be addressed; E-Mail:
[email protected]; Tel.: +86-0580-255-4852; Fax: +86-0580-255-4781. Received: 6 May 2014; in revised form: 13 May 2014 / Accepted: 21 May 2014 / Published: 23 May 2014
Abstract: We studied the expression of the non-metastatic clone 23 type 1 (nm23H1) gene, vascular endothelial growth factor (VEGF)-C, and its receptor VEGFR-3 using an in situ hybridization technique and immunohistochemical analyses with prostate cancer tissues and adjacent benign tissues of 52 human archival cases. The association between VEGF-C expression, microlymphatic count (MLC), and staining intensity for nm23H1 and VEGFR-3 was used to evaluate tumor metastasis and survival rate. MLC values were significantly higher in tumorous tissue than in non-cancerous tissue. VEGF-C mRNA, VEGFR-3, and nm23H1 were highly expressed in tumorous tissue. VEGFR-3 expression was greater in VEGF-C mRNA-positive tumors than in VEGF-C mRNA-negative tumors. The association of VEGFR-3 expression with VEGF-C mRNA and MLC suggested that the poor prognosis and tumor metastasis associated with VEGFR-3 expression may be due, in part, to its role in promoting angiogenesis. VEGF-C expression was significantly associated with tumor lymphangiogenesis, angiogenesis, and immune response as a potent multifunctional stimulating factor in prostate cancer. Expression of nm23H1 was significantly inversely correlated with lymph node metastasis. Furthermore, there was a strong negative correlation between the expression of nm23H1, VEGF-C mRNA, and MLC. These findings provide important information for prophylactic, diagnostic, and therapeutic strategies for prostate cancer. Keywords: human prostate cancer; VEGFR-3; VEGF-C; nm23H1
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1. Introduction Prostate cancer (PCa) is a prevalent, multifunctional, and heterogeneous disease, primarily affecting men over 50 years of age [1]. The incidence of PCa varies widely around the world, attributed to environmental, genetic, dietary, and hormonal factors, as well as their interactions [2–6]. Metastasis is the major cause of death from PCa, with the development of rich vascular networks of new lymphatic vessels and blood vessels in the tumor, namely angiogenesis, resulting in resistance to conventional therapies such as androgen ablation and cytotoxic chemotherapy [7]. A better understanding of the molecular mechanisms and genes involved in the initiation, progression, and metastasis of PCa, is necessary not only to help develop more effective therapeutic strategies, but also to advance basic cell science. It is important to elucidate the molecular mechanisms underlying the growth of new lymphatic vessels and the metastasis suppressor gene in human prostate tumors. The molecules that induce lymphatic vessel development, including members of the vascular endothelial growth factor (VEGF) family and their receptors (VEGF-A, VEGF-B, VEGF-C, VEGF-D, placental growth factor, VEGFR-1, VEGFR-2, and VEGFR-3), are associated with the angiogenesis induced by most cancer-cell types and certain tumor stromal cells [8–11]. Jackson et al. [12] first showed a widespread distribution of VEGF in PCa specimens and suggested that the VEGF165 and VEGF189 isoforms, novel 90- and 110-kD forms that are detected in the specimens, contribute to the establishment or progression of PCa. Ferrer et al. [13], Balbay et al. [14], Duque et al. [15], Strohmeyer et al. [16], and Mazzucchelli et al. [17] also reported increased levels of VEGF in PCa based on immunohistochemical findings. In addition, Krupski et al. [18], Jackson et al. [19], and Puyromaure et al. [20] studied the role of VEGF in the tissue-specific in vivo growth of PCa cells to examine its biologic impact on prostate tumors to promote angiogenesis and autocrine regulation of tumor growth, finding that VEGF acts as a multifunctional cytokine in prostate tumors and may have a prognostic impact in clinically-localized PCa. Furthermore, among the VEGF isoforms, Rinaldo et al. [21] reported an increase in VEGF-C expression in human PCa cells after androgen withdrawal. In general, VEGF-C interacts with VEGFR-2 to stimulate angiogenesis [22]. Another important lymphangiogenic factor, VEGFR-3, present in the endothelial cells of tumor blood vessels [23], promotes the metastasis of cancer cells via the lymphatic system [24]. In contrast, metastasis-suppressor genes in PCa, such as the nonmetastatic clone 23, maspin, HP1Hsα, and gelsolin genes, suppress the formation of spontaneous overt metastases [25–28]. Kauffman et al. [26] identified seven genes that suppress metastasis without affecting primary tumor growth: KAI1, CD44, mitogen activated protein kinase 4, nonmetastatic clone 23 type 1 (nm23H1), nm23H2, KiSS1, and BrMS1. Three of these genes (KAI1, CD44, and mitogen activated protein kinase 4) act as metastasis suppressor genes of PCa, while the others have yet to be tested in this cancer type. The nm23H1 gene is a putative tumor metastasis suppressor that might be associated with the expression of VEGF-C and its receptor. In the present study, we studied the expression of the nm23H1 gene, and VEGF-C and its receptor VEGFR-3 in PCa using an in situ hybridization technique. Immunohistochemical analyses using antibodies against nm23H1 and VEGFR-3 were also performed in PCa and adjacent benign tissues of 42 human archival cases in China. Furthermore, we examined the expression of VEGF-C mRNA, microlymphatic count (MLC), and the intensity of staining for nm23H1 and VEGFR-3 to evaluate tumor metastasis and the 5-year survival rate. Our aim was to
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reveal the expression of nm23H1, VEGF-C and its receptor VEGFR-3, and their association with PCa metastasis to elucidate the functional significance and mechanisms of nm23H1 and VEGF-C in PCa. These findings provide original data of Chinese PCa patients and important information for prophylactic, diagnostic, and therapeutic strategies for PCa. 2. Results and Discussion 2.1. Results VEGF-C hybridization was considered positive when the ratio of cancer cells whose cytoplasm was stained blue or purple black was greater than 10% (Figure 1). The adjacent benign tissue was not stained (Figure 2). Among the 42 specimens, VEGF-C mRNA expression was detected in 19 tumor specimens with a positive ratio of 45%, from which 7 patients were in TNM stages I and II, and 12 patients were in TNM stages III and IV (Table 1). In addition, VEGF-C mRNA expression was detected in 26% (8/31) of those negative for lymph node metastasis and 100% (11/11) in those positive for lymph node metastasis (p < 0.05; Table 2). Figure 1. Photomicrograph showing strong VEGF-C mRNA expression in cells of prostate cancer tissue, with positively-stained granules in the cytoplasm and nucleolus of tumor cells (in situ hybridization, magnification 400×).
VEGFR-3 was expressed mainly in the endothelial cells of lymphatic vessels, as indicated by brown or brown-yellow staining (Figure 3). VEGFR-3 positive lymphatic vessels were observed mainly in the stroma between PCa tissues. The microvessel walls had an irregular morphology and no new lymphatic vessels were observed in the center of the PCa tumor. MLC values were greater in PCa tissues (mean ± standard error, 8.61 ± 2.67/mm2) than in the adjacent benign tissues (4.51 ± 2.64/mm2; Table 2). The mean MLC values were 11.16 ± 1.39/mm2 for specimens positive for VEGF-C mRNA and 6.93 ± 1.80/mm2 for specimens negative VEGF-C mRNA. In addition, MLC values were higher in in stage III and IV PCa tissues or in patients with lymph node metastases than in stage I and II tumors or in patients without lymph node metastases (p < 0.05), or in the adjacent benign tissues (p < 0.01).
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The MLC of stage I and II PCa tissues was not significantly different than the MLC of the adjacent benign tissue. Consequently, there was no statistically significant relationship regarding the difference in the expression of VEGF-C mRNA and MLC in PCa tissues of different histopathologic grades. Figure 2. Photomicrograph showing negative staining of VEGF-C mRNA in cells of adjacent benign tissue (in situ hybridization, magnification 400×).
Table 1. Clinical characteristics of 42 PCa patients. Characteristics Age (years)
